Currently, no effective tool exists for screening or early diagnosis of head and neck squamous cell carcinoma (HNSCC). Here, we describe an approach for cancer detection based on analysis of patterns of serum immunoreactivity against a panel of biomarkers selected using microarray-based serologic profiling and specialized bioinformatics. We biopanned phage display libraries derived from three different HNSCC tissues to generate 5,133 selectively cloned tumor antigens. Based on their differential immunoreactivity on protein microarrays against serum immunoglobulins from 39 cancer and 41 control patients, we reduced the number of clones to 1,021. The performance of a neural network model (Multilayer Perceptron) for cancer classification on a data set of 80 HNSCC and 78 control samples was assessed using 10-fold cross-validation repeated 100 times. A panel of 130 clones was found to be adequate for building a classifier with sufficient sensitivity and specificity. Using these 130 markers on a completely new and independent set of 80 samples, an accuracy of 84.9% with sensitivity of 79.8% and specificity of 90.1% was achieved. Similar performance was achieved by reshuffling of the data set and by using other classification models. The performance of this classification approach represents a significant improvement over current diagnostic accuracy (sensitivity of 37% to 46% and specificity of 24%) in the primary care setting. The results shown here are promising and show the potential use of this approach toward eventual development of diagnostic assay with sufficient sensitivity and specificity suitable for detection of early-stage HNSCC in high-risk populations.